Both lefty-1 and lefty-2 genes are expressed on the left side of developing mouse embryos and are implicated in left-right (L-R) axis formation. With the use of transgenic analysis, the transcriptional regulatory regions of these genes responsible for their L-R asymmetric expression have now been investigated. The 9.5-kb upstream region of lefty-1 and the 5.5-kb upstream region of lefty-2 reproduced the expression pattern of the corresponding gene. Examination of deletion constructs revealed the presence of a left side-specific enhancer (ASE) that is essential and sufficient for lefty-2 asymmetric expression. In contrast, the asymmetric expression of lefty-1 was shown to be determined by a combination of bilateral enhancers and a right side-specific silencer (RSS). The 9.5-kb region of lefty-1 and the 5.5-kb region of lefty-2 responded to iv and inv, upstream genes of lefty-1 and lefty-2. The regulation of lefty-2 by iv and inv was mediated by ASE. These results suggest that, in spite of the similarities between lefty-1 and lefty-2, different regulatory mechanisms underlie their asymmetric expression.